Phytopathologia Mediterranea Firenze University Press The international journal of the www.fupress.com/pm Mediterranean Phytopathological Union

New or Unusual Disease Reports Characterization of Eutypa lata and Cytospora pistaciae causing dieback and canker of Citation: D. Aiello, G. Polizzi, G. Gusella, A. Fiorenza, V. Guarnaccia pistachio in Italy (2019) Characterization of Eutypa lata and Cytospora pistaciae causing die- back and canker of pistachio in Italy. Phytopathologia Mediterranea 58(3): Dalia AIELLO1,*, Giancarlo POLIZZI1, Giorgio GUSELLA1, Alberto 699-706. doi: 10.14601/Phyto-10880 FIORENZA1, Vladimiro GUARNACCIA2,3 Accepted: September 26, 2019 1 Dipartimento di Agricoltura, Alimentazione e Ambiente, sezione Patologia Vegetale, University of Catania, Via S. Sofia 100, 95123 Catania, Italy Published: December 30, 2019 2 Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Nether- Copyright: © 2019 D. Aiello, G. Poliz- lands 3 zi, G. Gusella, A. Fiorenza, V. Guar- DiSAFA, University of Torino, Largo Paolo Braccini, 2, 10095 Grugliasco, TO, Italy naccia. This is an open access, peer- *Corresponding author: [email protected] reviewed article published by Firenze University Press (http://www.fupress. com/pm) and distributed under the Summary. During the winter of 2017, dieback and canker symptoms were observed terms of the Creative Commons Attri- on pistachio (Pistacia vera) in two orchards in the Bronte area, Catania Province, Sic- bution License, which permits unre- ily, Southern Italy. Two different fungi were consistently isolated from infected tissues. stricted use, distribution, and reproduc- Morphological observations and multi-locus phylogenies using five genomic loci (ITS, tion in any medium, provided the origi- act, rpb2, tef1 and tub2) identified these fungi as Cytospora pistaciae and Eutypa lata. nal author and source are credited. Pathogenicity tests on 5-y-old potted plants of P. vera grafted on terebinth P.( terebin- Data Availability Statement: All rel- thus) reproduced similar symptoms as those observed in nature, and Koch’s postulates evant data are within the paper and its were fulfilled for these two pathogens. This study is the first to report dieback and can- Supporting Information files. ker diseases of pistachio caused by C. pistaciae and E. lata in Italy.

Competing Interests: The Author(s) Keywords. Pathogenicity, molecular analysis, disease symptoms, Pistacia vera. declare(s) no conflict of interest.

Editor: Lizel Mostert, Faculty of AgriSciences, Stellenbosch, South Africa. INTRODUCTION

Pistachio is cultivated in the southern regions of Italy, of which Sicily is the main production area. The province of Catania (with 430 ha of pista- chio), followed by the provinces of Caltanissetta (with 220 ha) and Agrigento (with 145 ha) are the largest pistachio-producing areas, with a total produc- tion of 3,878 tons (AGRISTAT, 2017). Currently, the commune of Bronte in Catania Province represents the most important area of Sicily for pistachio production, and pistachio is an important economic resource for this terri- tory (Barone and Marra, 2004). In this area, different pistachio cultivars are grafted on terebinth plants which are grown on volcanic soils (Barone et al., 1985). Few studies have been conducted to investigate pistachio diseases occurring in Italy, and only a few diseases have been reported to date. These include branch dieback (caused by Botryodiplodia sp.), leaf spot (Alternaria alternata), anthracnose, branch and twig cankers (Botryosphaeria dothidea)

Phytopathologia Mediterranea 58(3): 699-706, 2019 ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phyto-10880 700 Dalia Aiello et alii and phylloptosis and leaf spots (mainly caused by Septo- DNA extraction, PCR amplification and sequencing ria pistaciae) (Casalicchio, 1963; Schilirò and Privitera, 1988; Frisullo et al., 1996; Vitale et al., 2007). In east- Extractions of genomic DNA were performed from ern Sicily, cankers and decline caused by Liberomyces pure cultures, as reported elsewhere (Guarnaccia and pistaciae Voglmayr, Vitale, Aiello, Guarnaccia, Luongo Crous, 2017), using the Wizard Genomic DNA Purifica- & Belisario are the most important pistachio diseases tion Kit (Promega Corporation). Partial regions of five (Vitale et al., 2018). Blight caused by Arthrinium xeno- loci were amplified. The primers ITS5 and ITS4 (White cordella Crous was also recently reported on pistachio et al., 1990) were used to amplify the internal tran- fruit in the Agrigento Province (Aiello et al., 2018). scribed spacer region (ITS) of the nuclear ribosomal During the winter of 2017, pistachio trees with die- RNA operon, including the 3’ end of the 18S rRNA, the back, canker and gummosis symptoms were observed first internal transcribed spacer region, the 5.8S rRNA in the area of Bronte. Following culturing from necrot- gene; the second internal transcribed spacer region and ic tissues, two fungal species were consistently isolated. the 5’ end of the 28S rRNA gene. The primers ACT- Cankers from one orchard generated colonies of Cyto- 512F and ACT-783R (Carbone and Kohn, 1999) were spora while cankers from a second orchard generated used to amplify part of the actin gene (act). The partial Eutypa colonies. beta-tubulin (tub2) gene was amplified with primers Bt- The aim of the present study was to investigate the 2a and Bt-2b (Glass and Donaldson, 1995). The prim- etiology of pistachio canker diseases, which could repre- ers EF1-728F and EF1-986R (Carbone and Kohn, 1999) sent new threats for the pistachio production of Sicily. were used to amplify part of the translation elongation factor 1-α gene (tef1). The primers 5f2/7cr were used to amplify part of rpb2 (O’Donnell et al., 2010). The regions MATERIALS AND METHODS ITS, act, tef1 and rpb2 were amplified for the species of Cytospora using the PCR programmes adopted by Law- Isolation and morphology of fungi rence et al. (2018) and Jami et al. (2018). The regions ITS and tub2 were amplified for the species of Eutypa follow- Surveys were conducted in ten pistachio orchards ing the PCR programmes used by Moyo et al. (2018a). with histories of branch canker and dieback in east- The PCR products were sequenced in both directions ern Sicily (Catania Province). Approximately 20 using the BigDye® Terminator v. 3.1 Cycle Sequencing symptomatic pistachio branches with canker were col- Kit (Applied Biosystems Life Technologies), after which lected from each orchard for analyses. Sub-cortical amplicons were purified through Sephadex G-50 Fine and wood fragments (about 5 × 5 mm) were cut from columns (GE Healthcare) in MultiScreen HV plates the margins between affected and healthy branch tis- (Millipore). Purified sequence reactions were analyzed sues. Tissue pieces were disinfected in 1.2% sodium on an Applied Biosystems 3730xl DNA Analyzer (Life hypochlorite for 60 s, rinsed in sterile water and Technologies). The DNA sequences generated were ana- dried on sterile filter paper. The fragments were then lyzed and consensus sequences were computed using the placed into Petri plates containing potato dextrose program SeqMan Pro (DNASTAR). agar (PDA, Oxoid) amended with 100 mg L-1 of strep- tomycin sulfate (Sigma-Aldrich), and incubated at Phylogenetic analyses room temperature (25 ± 5°C). Fungal colonies consist- ently growing from symptomatic tissues were cultured Novel sequences generated in this study were blast- into new PDA plates. To obtain pure cultures, single- ed against the NCBIs GenBank nucleotide database, to conidium or hyphal-tip isolations were performed determine the closest relatives to be included in the phy- after 1 month incubation at room temperature under logenetic analyses. Blast analyses indicated that three natural light conditions. Isolates for each putative isolates belonged to Cytospora and the remaining four fungal pathogen (four isolates of Eutypa and three to Eutypa. Sequence alignments of the different gene of Cytospora) were characterized by morphological, regions, including sequences obtained from this study molecular and phylogenetic analyses (Table 1). These and sequences from GenBank, were initially performed cultures were deposited in the working collection of using the MAFFT v. 7 online server (http://mafft.cbrc. Dr Pedro Crous (CPC), at the Westerdijk Fungal Bio- jp/alignment/server/index. html) (Katoh and Stand- diversity Institute, Utrecht, The Netherlands (Table 1). ley, 2013), and then manually adjusted in MEGA v. 7 Size and shape of conidia were recorded for each fun- (Kumar et al., 2016). To establish the identity of the fun- gal isolate grown on PDA for 2 weeks at 25 ± 1°C. gal isolates, phylogenetic analyses were conducted using Diseases of pistachio in Italy 701 one locus (data not shown) as well as concatenated anal- showed dieback. Under the bark of affected branch- yses of four loci (ITS, act, tef1 and rpb2) for Cytospora es, cankers were characterized by discolouration and spp. and two loci (ITS and tub2) for Eutypa spp., as indi- necrosis, and in some cases discolouration extended to cated by blast analysis. Additional reference sequences the vascular tissue (xylem) and pith. Two different fun- were selected based on recent studies on Cytospora and gal colony types were consistently obtained from isola- Eutypa species (Lawrence et al., 2018, Moyo et al., 2018a, tions from symptomatic tissues (Figure 1) taken from b). Phylogenetic analyses were based on Maximum the two orchards. Cankers from one orchard generated Parsimony (MP) for all the individual loci and for the Cytospora colonies while cankers from the other orchard multi-locus analyses. The MP analyses were carried out generated Eutypa colonies. The same symptoms in the using PAUP (Swofford, 2003). Phylogenetic relationships remaining orchards investigated in the Bronte area pro- were estimated by heuristic searches with 100 random duced colonies of L. pistaciae (Vitale et al., 2018). addition sequences. Tree bisection-reconnection was Conidia of three representative isolates of Cytospora used, with the branch swapping option set on ‘best trees’ were in accordance with the description by Lawrence only, with all characters weighted equally and alignment et al. (2018) of C. pistaciae Lawr., Holland & Trouillas. gaps treated as fifth state. Tree length (TL), consistency The four MP trees derived from the single gene sequence index (CI), retention index (RI) and rescaled consistence alignments (ITS, act, tef1 and rpb2) were topologically index (RC) were calculated for parsimony and the boot- similar, confirming that the three isolates used for the strap analyses (Hillis and Bull 1993) were based on 1,000 molecular analyses were Cytospora. The combined phy- replicates. Sequences generated in this study were depos- logeny of Cytospora species consisted of 35 sequences, ited in GenBank (Table 1). including the outgroup sequences of Diaporthe limoni- cola (culture CBS 142549; Guarnaccia and Crous, 2017). A total of 2,056 characters (ITS: 1–574, act: 581–890, Pathogenicity of representative isolates tef1: 897–1289, rpb2: 1296–2056) were included in the Pathogenicity tests with one representative isolate of phylogenetic analysis of Cytospora spp. For the phylog- C. pistaciae (CPC34208) and one of E. lata (CPC34213; eny of Cytospora species, 489 characters were parsimo- Table 1) were carried out to satisfy Koch’s postulates. ny-informative, 336 were variable and parsimony-unin- These tests were carried out in a growth chamber main- formative and 1,213 characters were constant. A maxi- tained at 25 ± 1°C. Potted 5-y-old plants of P. vera mum of 1,000 equally most parsimonious trees were grafted onto P. terebinthus were used for artificial inoc- saved (Tree length = 1 552, CI = 0.743, RI = 0.782 and ulations. Three plants were inoculated with each iso- RC = 0.581). Bootstrap support values from the parsi- late. Six wounds were made on individual plant stems mony analysis were plotted on the phylogenetic trees approx. 8-10 cm apart from each other. presented in Figure 2. In the combined analyses, the Inoculations were made on stems after removing of three representative isolates clustered with four refer- bark discs with a cork borer, placing a 5 mm plug from ence strains of C. pistaciae. The individual alignments a 14-d-old PDA culture of test isolate into the wound and trees of the single loci used in the analyses were and covering with Parafilm® (Pechney Plastic Packaging compared with respect to their performance in species Inc.) to prevent desiccation. An equivalent number of recognition. Cytospora pistaciae was differentiated and plants and inoculation sites were inoculated with ster- identified in all single-gene analyses. ile PDA plugs to serve as controls. The inoculated plants Cytospora terebinthi Bres. has been reported in Italy were observed once each month for symptoms develop- as the causal agent of cankers and gummosis of pista- ment, and a final assessment was conducted 5 months chio (Corazza et al., 1990; Furnitto, 1984), while other after inoculation. To fulfil Koch’s postulates, re-isola- Cytospora species have been reported in other crops, tions were carried out following the procedure described including peach (Hampson and Sinclair., 1973; Banko above, where tissue fragments were plated onto PDA. and Helton, 1974). The of Cytospora species Each re-isolated was identified through morpho- associated with fruit and nut crops was recently revised, logical characteristics. and C. pistaciae was described as a new species on pista- chio in California, but the pathogenicity of this species was not investigated (Lawrence et al. 2018). RESULTS AND DISCUSSION Conidia of four isolates of Eutypa were in accord- ance with the description of E. lata by Moyo et al. Symptomatic plants showed cankers with crack- (2018b). The two MP trees derived from the single gene ing and gum exudation, and often branches or shoots sequence alignments (ITS and tub2) were topologi- 702 Dalia Aiello et alii cally similar, and this confirmed that the four isolates Eutypa lata is a pathogen with a wide host range, used in this study were Eutypa. All the species belong- occurring in more than 160 hosts (Farr and Rossman, ing to Eutypa and other used in the multi- 2017). In Italy, E. lata has been reported on Acer sp. in locus phylogeny consisted of 29 sequences with the out- Sicily (Greuter et al., 1991), Ribes rubrum (Prodorutti et group sequences of L. pistaciae (CBS 144255; Vitale et al., 2008), olive trees (Tosi and Natalini, 2009) and Vitis al., 2018). A total of 1,076 characters (ITS: 1–582, tub2: vinifera (Acero et al., 2004). Eutypa dieback and gum- 589–1,076) were used for the Xylariales analysis, and mosis of pistachio caused by E. lata has been reported 453 characters were parsimony-informative, 166 were only in Greece (Rumbos, 1986). variable and parsimony-uninformative and 451 charac- Five months after artificial inoculation, symptoms ters were constant. A maximum of 1,000 equally most produced from each fungus in trees were similar to those parsimonious trees were saved (Tree length = 1 669, CI present on trees in the field. These consisted of external = 0.648, RI = 0.786 and RC = 0.509). Bootstrap support cankers and gumosis produced around the inoculation values from the parsimony analysis were plotted on the sites, with small cracks present in each sunken lesion. phylogenetic trees presented in Figure 3. In the com- After removing the bark, a dark discolouration and bined analyses, the four isolates were related to refer- necrotic tissues were visible (Figure 1). The respective ence isolates of E. lata. The individual alignments and inoculated pathogens were re-isolated from symptomatic trees of the single loci used in the analyses were com- tissues, thus fulfilling Koch’s postulates. No symptoms pared with respect to their performance in species rec- were observed on control (uninoculated) plants. ognition. Eutypa lata was differentiated and identified This is the first report of E. lata and C. pistaciae in all single-gene analyses. associated with cankers on pistachio in Europe. Further

Figure 1. Symptoms reproduced from mycelial plug inoculation with Cytospora pistaciae (a) and Eutypa lata (b) on 5-y-old potted plants of Pistacia vera 5 months after inoculation with respective fungi. Cultural characteristics of Cytospora pistaciae (c) and Eutypa lata (d) colonies grown on PDA are also illustrated. Diseases of pistachio in Italy 703 ------tub2 (Continued) ------rpb2 KY417808 KY417805 KU710976 KU710960 KU710953 MN078082 MN078081 MN078080 a ------tef1 MN078079 MN078078 MN078077 MG971654 MG971654 MG971516 MG971517 MG971517 MG971518 MG971518 MG971515 MG971515 MG971519 MG971520 MG971521 MG971522 MG971522 MG971605 MG971605 MG971514 MG971514 MG971630 MG971630 MG971645 MG971645 GenBank Number ------act KU711006 KU711000 MN078065 MN078064 MN078063 MG972091 MG972091 MG971951 MG971952 MG971953 MG971953 MG971950 MG971950 MG971954 MG971955 MG971956 MG971957 MG971957 MG972044 MG972044 MG971949 MG971949 MG972069 MG972069 MG972083 MG972083 ITS KF765671 KF923248 KF923249 KR045645 AF260266 AY347358 AY347358 AY347327 AY347374 AY347374 AY347368 AY347368 KY417740 AY347377 AY347377 KR045634 AY347350 AY347350 KY417737 KR045624 NR137542 MN078068 MN078067 MN078066 MG971943 MG971943 MG971801 MG971801 MG971802 MG971802 MG971803 MG971803 MG971800 MG971800 MG971804 MG971805 MG971806 MG971807 MG971807 MG971895 MG971895 MG971799 MG971799 MG971920 MG971920 MG971935 MG971935 Locality China USA Italy Italy Italy California, USA California, USA California, USA California, USA Russia Russia California, USA California, USA California, USA California, USA Russia Russia China USA China USA Zimbabwe Zimbabwe Entebbe, Uganda Entebbe, Tasmania, Australia Australia Tasmania, India Uruguay Australia Australia Chile China California, USA California, USA California, USA Australia Australia Host Juglans regia Juglans Punica granatum Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Malus domesticaMalus Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Salix acutifolia acutifolia Salix Sorbus pohuashanensis Populus deltoides Salix psammophila psammophila Salix Punica granatum Eucalyptus camaldulensis Eucalyptus camaldulensis Eucalyptus grandis Eucalyptus nitens Eucalyptus Eriobotrya japonica Eriobotrya japonica Eucalyptus grandis Eucalyptus globulus globulus Eucalyptus Eucalyptus globulus globulus Eucalyptus Prunus cerasifera Prunus Pistacia vera vera Pistacia Juglans regia regia Juglans Eucalyptus globulus Eucalyptus pauciflora Eucalyptus Culture No. Culture CFCC 89624 5A-80 = CBS 144244 5A-80 CPC 34211 CPC 34209 CPC 34208 = CBS 144226 KARE444 KARE443 = CBS 144238 KARE443 KARE442 KARE441 KARE441 MFLUCC 15–0507 MFLUCC KARE270 = CBS 144506 KARE270 KARE269 KARE268 KARE232 MFLUCC 15–0860 MFLUCC CFCC 50015 CBS 144235 CFCC 89634 CBS 144237 CMW 40048 CMW 40051 CMW 5309 ATCC 96150 ATCC IMI136523 CMW 6509 CMW 8549 CMW 5700 CFCC 89956 KARE264 9c–24 = CBS 144234 StanfordT3T StanfordT3T CMW 6735 C. sacculus C. sacculus C. punicae C. punicae C. pistaciae C. parasitica C. parasitica C. parapistaciae C. nivea C. leucostoma C. leucostoma C. joaquinensis C. gigaspora C. gigaspora C. granati C. eucalypticola C. eucalypticola C. eriobotryae C. eriobotryae C. disciformis C. disciformis C. diatrypelloidea C. cinereostroma C. cinereostroma C. cincta C. cincta C. californica C. berkeleyi Cytospora austromontana Collection details and GenBank accession numbers for isolates included in this study. included isolates for numbers 1. Collection GenBank accession and details Table Species 704 Dalia Aiello et alii ------tub2 KY111608 KY111588 KR363998 KT425167 KY752794 KY752791 GQ294029 DQ006999 DQ006960 DQ006967 HQ692485 HQ692485 HQ692501 HQ692501 HQ692499 HQ692499 HQ692493 HQ692493 MN078076 MN078075 MN078074 MN078073 MH316764 MH316763 MH316765 MH797697 ------rpb2 KY417814 MH797629 a ------tef1 MF418501 GenBank Number ------act ITS AJ302465 AJ302465 AJ302462 AJ302462 AJ302467 AJ302467 AY684224 KY111629 KY111634 KT425232 KY752766 KY752762 KY417746 MF959502 GQ293962 KU982636 MF418422 DQ006943 DQ006923 HQ692608 HQ692608 HQ692611 HQ692611 HQ692615 HQ692615 HQ692610 HQ692610 KM396647 KM396646 MG873478 MG873477 MN078072 MN078071 MG873479 MG873479 MN078070 MN078069 KM396615 MH797562 Locality Thailand France France Thailand Thailand Spain Brazil Thailand Brazil France Italy USA South AfricaSouth South AfricaSouth France Switzerland Australia Australia Italy Italy Italy Italy Australia Australia Australia Australia Australia Australia South AfricaSouth South AfricaSouth Brazil Malta Norway Russia Russia Russia Russia . Host sp unknown Robinia pseudoacacia Robinia Robinia pseudoacacia Robinia unknown Hevea brasiliensis Hevea Atriplex halimus unknown unknown unknown Arthrocnemum fruticosum fruticosum Arthrocnemum Pistacia vera vera Pistacia Vitis vinifera vinifera Vitis Vitis vinifera vinifera Vitis Vitis vinifera vinifera Vitis Sarothamnus scoparius Acer pseudoplatanus pseudoplatanus Acer Ficus macrophylla macrophylla Ficus Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Pistacia vera vera Pistacia Vitis vinifera vinifera Vitis Ceanothus Schinus molle Prunus armeniaca Prunus salicina unknown Citrus limon Salix borealis borealis Salix Salix alba alba Salix Salix alba alba Salix Culture No. Culture MFLUCC 17–2143 MFLUCC CBS 242.87 DFMAL100 DFMAL100 MFLUCC 17–2142 MFLUCC MFLUCC 17–0371 MFLUCC F-092,373 F-092,373 HUEFS 192196 MFLUCC 17–2144 MFLUCC HUEFS 136877 CBS 250.87 CBS 144225 MSUELM13 MSUELM13 STEU 8107 STEU STEU 8098 STEU CBS 284.87 CBS 219.87 ADFIC100 ADFIC100 CPC 34216 CPC 34215 CPC 34214 CPC 34213 EP18 SACEA01 SACEA01 ADSC300 CBS 121430 CBS 120837 HUEFS 194228 CBS 142549 CBS 240.87 MFLUCC 14–1052 MFLUCC MFLUCC 16–0509 MFLUCC ITS: internal transcribed spacers 1 and 2 together with 5.8S nrDNA; act: actin; tef1: translation elongation factor 1-α gene; rpb2: RNA polymerase second largest subunit; tub2: beta- tub2: subunit; largest second polymerase rpb2: RNA 1-α gene; factor elongation act: actin; translation tef1: 5.8S nrDNA; with 2 together transcribed 1 and ITS: spacers internal

Peroneutypa scoparia scoparia Peroneutypa Peroneutypa rubiformis rubiformis Peroneutypa Peroneutypa longiasca longiasca Peroneutypa Peroneutypa kochiana kochiana Peroneutypa Peroneutypa diminutispora diminutispora Peroneutypa Peroneutypa diminutiasca diminutiasca Peroneutypa Peroneutypa curvispora curvispora Peroneutypa Peroneutypa alsophila alsophila Peroneutypa Liberomyces pistaciaeLiberomyces Eutypella vitis vitis Eutypella Eutypella microtheca microtheca Eutypella Eutypella citricola citricola Eutypella Eutypa tetragona tetragona Eutypa Eutypa maura Eutypa leptoplaca Eutypa lata Eutypa cremea Eutypa Diatrypella atlantica Diatrypella atlantica Diaporthe limonicola Diaporthe limonicola Cryptosphaeria subcutanea C. salicicola C. salicicola tubulin. Sequences generated in this study indicated in italics. Ex-type and ex-epitype cultures are indicated in bold. indicated are cultures Ex-type in italics. ex-epitype and indicated in this study Sequences generated tubulin. C. salicacearum Table 1. (Continued). Table Species a Diseases of pistachio in Italy 705

Figure 3. The first of four equally most parsimonious trees obtained from a heuristic search of the combined ITS, and tub2 sequence Figure 2. The first of two equally most parsimonious trees obtained alignments of species belonging to Eutypa and other genera of Dia- from a heuristic search of the combined ITS, act, tef1 and rpb2 trypaceae. Bootstrap support values are shown at the nodes. The sequence alignments of Cytospora spp. Bootstrap support values are strains isolated in this study are shown in red and the scale bar rep- shown at the nodes. The strains isolated in this study are shown in resents the number of changes. The tree was rooted to Liberomyces red and the scale bar represents the number of changes. The tree pistaciae (CBS 144255). was rooted to Diaporthe limonicola (CBS 142549). studies should investigate the role of propagation mate- Barone E., Marra F.P., 2004. “The Pistachio Industry in rial, mechanical injuries and pruning wounds in disease Italy: current situation and prospects”. Nucis 12: 16–19. transmission and spread. Barone E., Caruso T., Di Marco L., 1985. Il pistacchio in Sicilia: superfici coltivate e aspetti agronomi- ci. L’Informatore Agrario 40: 35–42. ACKNOWLEDGEMENTS Carbone I., Kohn L.M., 1999. A method for designing primer sets for the speciation studies in filamentous This research was funded by Research Project 2016- ascomycetes. Mycologia 91: 553–556. 2018 “Emergent Pests and Pathogens and Relative sus- Casalicchio G., 1963. Le avversità del pistacchio (Pistacia tainable Management Strategies”, financed by University vera). Informatore Fitopatologico 13: 148–188. of Catania. Corazza L., et al., 1990. Principaux aspects phyto- pathologiques del la pistache en Italie. Programme de recherche Agrimed Amélioration génétique de deux LITERATURE CITED espèces de fruits secs méditerranéens: l’amandier et le pistachier: 319. Acero F.J., Gonzalez V., Sanchez-Ballesteros J., Rubio V., Farr D.F., Rossman A.Y., 2017. Fungal Databases, Syst. Checa J., … Pelaez F., 2004. Molecular phylogenetic Mycol. Microbiol. Lab., ARS, USDA. Retrieved from studies on the based on rDNA-ITS https://nt.ars-grin.gov/fungaldatabases/. sequences. Mycologia 96: 249–259. Frisullo S., Lops F., Carlucci A., Camele I., 1996. Par- Aiello D., Gulisano S., Gusella G., Polizzi G.,Guarnaccia assiti fungini delle piante dell’Italia meridionale. V., 2018. First report of fruit blight caused by L’antracnosi del pistacchio. Informatore Fitopatologico Arthrinium xenocordella on Pistacia vera in Italy. 46: 45–47. Plant Disease 102: 1853. Furnitto S., 1984. Un deperimento di piante di pistac- AGRISTAT, 2017. AGRISTAT (Online). Available: http://agri. chio associato a cancri di Cytospora terebinthi. Tesi istat.it/sag_is_ pdwout/jsp/dawinci. jsp?q=plC190000030 sperimentale di laurea, Anno accademico 1983-84, 000203200&an=2017&ig=1&ct=270&id=15A|21A|30A. Istituto di patologia vegetale, Facoltà di agraria di Banko T.J., Helton A.W., 1974. Cytospora induced chang- Catania. es in stems of Prunus persica. Phytopathology 64: Glass N.L., Donaldson G.C., 1995. Development of prim- 899–901. er sets designed for use with the PCR to amplify con- 706 Dalia Aiello et alii

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